This invention relates to separation and purification of polyelectrolytes. In particular, the present invention relates to the purification of biochemical materials such as proteins and more particularly nucleic acids.
Purification of molecular species constitutes a crucial part to their production and utility. This is particularly important in the biotechnology and diagnostics fields. The present invention describes polymeric separation media and methods useful for the purification of molecular species that are polyelectrolytes, such as proteins and nucleic acids. The terms nucleic acid and polynucleotide are used interchangeably, and are used here to signify either a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Unless otherwise specified, the terms polynucleotide and oligonucleotide are used interchangeably.
There is a large body of publications dealing with synthesis, functionalization, and use of ion-exchangers for chromatography and biomolecule purification. (The following patents are herein incorporated by reference in their entirety.) BACKUS et al (U.S. Pat. Nos. 5,582,988 and 5,622,822, and CA 2,157,968) describes a method for isolation of nucleic acids from a lysate by contacting with polymers containing basic groups, such as polyethyleneimine. However, an acidic medium was required for binding, and a strong alkali and heat were found to be the agents most successful in releasing, or eluting, the nucleic acids from the polymer.
COLPAN et al (DE patent number 4139664) describe a method for isolating nucleic acid from cellsxe2x80x94by lysis of the cells, then elution of nucleic acid fixed to separation medium surface. However, the method recovers the nucleic acids by eluting with a buffer of high ionic strength.
HENCO et al (DE 3639949) describes a method for the Separation of long-chain nucleic acidsxe2x80x94using a porous separation medium to fix the nucleic acids. The invention uses selective salt elution to first wash off the short chain nucleic acids whereas the long-chain nucleic acids are subsequently removed from the anion exchanger using a washing solution of high ionic strength.
SELIGSON and SHRAWDER (U.S. Pat. No. 4,935,342) describe the classical method of isolation of nucleic acids by chromatography on anion exchanger using salt gradients. After the nucleic acids become bound to the ion exchange material and washed, the bound nucleic acids are eluted by passing through the column a salt solution of high molarity.
GANNON (EP 366438) describes the separation of nucleic acid from protein by contacting with cation exchanger at pH below isoelectric point of a protein, i.e., it binds the proteins not the DNA.
Similar principles are offered in U.S. Pat. No. 3,433,782, where selective elution steps effected with varied molarities of LiCLO4 or NaClO4.
Similar principles are offered in U.S. Pat. No. 434,324, where purification of deoxyribonucleic acid is accomplished by using anion exchange material, washing with weak ionic salt solution and elution with strong ionic salt solution.
Similar principles are offered by Bourque and Cohen (WO 9514087), where detection of charged oligonucleotides is accomplished by adsorbing on an ion exchange resin, eluting with a high salt buffer and detection. The oligonucleotides bind to the anion exchange resin at 40-65xc2x0 C., while the desorbing from the resin with a high salt buffer is performed at 40-65xc2x0C.
BRUCE et al (Patent Number WO 9411103; GB 9223334) describes magnetizable polymer-based particles derived with ligand having direct binding affinity for nucleic acids etc., for the separation of nucleic acids. The polymer is agarose. The ligand is one capable of assuming a positive charge at pH 7 or below, and is capable of reversibly binding directly to a negatively charged group or moiety in the target molecules. The selected ligands are amines, such as dimethylaminoethyl, and triethanolamine; all have a pKa higher than in our invention.
ADRIAANSE et al (EP 389063 A) describe a method that is widely adapted as diagnostics kits in the art, namely the isolation of nucleic acid using chaotropic agents for nucleic acid binding to solid phase. The use of high concentration of chaotropes, e.g., guanidinium thiocyanate, forces the DNA to precipitate and interact with many surfaces. The present invention avoids the use of highly concentrated chaotropes during purification.
NELSON et al (EP 281390) offered a method for the separation of small nucleotides from larger ones by binding to polycationic supportxe2x80x94which does not retain smaller sequences for hybridization assays. The bound nucleic acids were apparently eluted, if needed, by 50% formamide, or other salts.
JP 06335380 A describes a carrier for bonding nucleic acid, which has hybrid-forming base sequence fixed over surface of insoluble solid fine particles. The base of binding is the interaction between complimentary sequences of polynucleotides.
U.S. Pat. No. 4,672,040 also describes a silanized magnetic particles, for use in nucleic acid hybridization, where the principle of binding is the interaction between the hybrid-forming base sequence fixed over surface of insoluble solid fine particles.
MACFARLANE (U.S. Pat. No. 5,300,635), uses quaternary amine surfactantsxe2x80x94for isolating nucleic acids from a biological sample by forming complexes which can be dissociated.
HILL (WO 8605815) also describes the use of magnetized nucleic acid sequence comprising single or double-stranded nucleic acid linked to magnetic or magnetizable substance.
HORNES and KORSNES (U.S. Pat. No. 5,512,439) also describe the detection and quantitative determination of target RNA or DNAxe2x80x94by contacting sample with magnetic particles carrying 5xe2x80x2-attached DNA probe.
REEVE (U.S. Pat. No. 5,523,231) describes a method of making a product solution containing a nucleic acid by treating a starting solution containing the nucleic acid by the use of suspended magnetically attractable beads which do not specifically bind the nucleic acid, by precipitating the nucleic acid out of the starting solution in the presence of the suspended magnetic beads whereby a nucleic acid precipitate becomes aggregated with and entraps the beads, followed by separating the precipitate and the entrapped beads and adding a liquid to the precipitate and the entrapped beads to re-dissolve the nucleic acid and re-suspend the beads.
The present invention is directed to polymeric separation media and to methods useful for the purification of polyelectrolytes, particularly polynucleotides.
In contrast to the prior art methods, the present invention provides mild conditions that avoid the unfavorable, and sometimes harsh conditions otherwise required to bind and elute biomolecules in related art. As mentioned above, prior nucleic acid isolation or purification methods include steps such as heating, and reagents such as strong alkalis, or highly concentrated salts and chaotropes. In addition to being automation and operator unfriendly, these steps/reagents require additional efforts to implement, neutralize, or remove.
The polymeric separation media possess multiple pendant groups, i.e., functional groups, whose protonation state is pH-dependent. Consequently, the amount of charge created on the separation medium can be controlled by adjustment to the pH of the buffer solution where the polymeric separation medium is suspended. As an example, if the pendant groups are basic (B) and possess a pKa of 7, then, at neutral pH (i.e., pH=7), 50% of the groups will acquire positive charges (BH+). Since the pH scale is logarithmic, then, at pH=8, the percent of the positively charged groups will drop to 10%, and to 1% at pH=9. Similarly, if the basic groups possess a pKa of 6, then at pH=9, only 0.1% of the groups will be positively charged, and so forth.
It is an aspect of this invention that at neutral pH, basic groups with pKa as stated above can bind, preferentially strongly, to polyelectrolytes with high negative charge density, such as polynucleotides. At low amount of the positive charges, the separation medium will bind preferably to polyelectrolytes with the most negative charges, such as DNA. Other polyelectrolytes such as proteins, even though they may possess negative charges, will not bind as strongly. The difference in binding strength is attributed to the nature of the charge distribution on polynucleotides and proteins, with polynucleotides possessing multiple, repeated negative charges. As the polymeric separation medium possesses multiplicity of positive charges, even when not totally protonated, multiple electrical interaction (attraction) can occur with the multiple negative charges on the polynucleotides. Bound proteins can be washed off the separation medium by the choice of the pH of the wash buffer. Elution may also be carried in multiple steps, each with an increasing pH, where proteins would elute first, while DNA elutes last. In a preferred embodiment, a negatively charged polyelectrolyte binds to the separation medium at pH 7, as discussed above, and can be eluted in steps with change of the pH to 8 to 10, thus washing off the proteins at pH 8, while eluting DNA at the higher pH. In the above scheme, DNA and protein molecules can be selectively separated form a mixture thereof.
It is an important aspect of this invention, and one of its most preferred embodiments to purify nucleic acids, that the binding step of the positively charged polymeric separation medium occur at a neutral pH where the separation medium is not maximally charged. If the separation medium carries a high positive charge density, then the binding to the highly negatively charged DNA would be too strong to be separated without recourse to harsh conditions, as observed in the prior art listed above.
A purification method using such a separation medium is capable of discriminating between the binding ability of the pH-dependent groups to the different solutes present in a mixture. In addition to selectively binding polyelectrolytes, the separation medium can also selectively elute polyelectrolytes.
Toward operating under mild conditions, an aspect of the present invention is that the pendant groups carry a certain amount of positive charges at neutral pH. It is clear from the discussion above that pendant groups with pKa of about 6 to 7 can bind polynucleotides, and quite strongly at neutral pH. As such, the binding step can thus be accomplished at neutral pH.
In a preferred embodiment, the polymeric separation medium is water-insoluble. In another preferred embodiment, the polymeric separation medium encapsulates a magnetic core, so that it can be separated by the application of a magnetic field.
In another preferred embodiment the polymeric separation medium contains pendant groups that exhibit pKa values of less that about 7, and the medium (buffer) containing the polymeric separation medium is buffered so that the pH of the buffer has a neutral value, thus causing the pendant groups of the polymeric separation medium to be not completely protonated (positively charged) at pH of at least 7.
Preferred pendant basic groups are derived from a base which is a member of the group consisting of pyridine, quinoline, imidazole, and pyrimidine.
Preferred pendant acidic groups are members of the group consisting of a carboxylic group and a phenolic group.